LNA-G Process

ABSTRACT

Recent advancements in LNA oligonucleotides include the use of amine linkers to link an LNA antisense oligonucleotide to a conjugate group. For example please see WO2014/118267. The present invention originates from the identification of a problem when de-protecting LNA oligonucleotides which comprise an aliphatic amine group and DMF protected LNA G nucleoside, which results in the production of a +28 Da impurity. This problem is solved by using acyl protection groups on the exocyclic nitrogen of the LNA-G residue, rather than the standard DMF protection group.

FIELD OF THE INVENTION

The present invention relates to the field of LNA antisenseoligonucleotide conjugates and to methods of synthesis thereof.

BACKGROUND TO THE INVENTION

Recent advancements in LNA oligonucleotides include the use of aminelinkers to link an LNA antisense oligonucleotide to a conjugate group.For example please see WO2014/118267. The present invention originatesfrom the identification of a problem when de-protecting LNAoligonucleotides which comprise an aliphatic amine group and DMFprotected LNA G nucleoside, which results in the production of a +28 Daimpurity. This problem is solved by using acyl protection groups on theexocyclic nitrogen of the LNA-G residue, rather than the standard DMFprotection group.

STATEMENT OF INVENTION

The invention provides for a method of preparing a LNA oligonucleotidecomprising the steps of:

a) Incorporating at least one LNA-G monomer comprising an acyl protectedexocyclic nitrogen into an oligonucleotide;

b) Incorporating at least one optionally protected aliphatic amine groupinto the oligonucleotide;

c) deprotecting the acyl protected exocyclic nitrogen of the at leastone LNA-G monomer by removal of the acyl protection group;

-   -   wherein steps a) and b) can occur in either order.    -   Suitably, when present, other G monomers, e.g. DNA G-monomers        incorporated into the LNA oligonucleotide, they are also acyl        protected on their exocyclic nitrogen, for example by a step of        incorporating G-monomer, e.g. a DNA G-monomer or a 2′substituted        G-monomer (e.g. 2′-O-methoxyethyl G-monomer, or a 2′methyl G        monomer), wherein the G monomer comprises an acyl protected        exocyclic nitrogen into an oligonucleotide, e.g. using a acyl        protection group as described herein, e.g. as defined by the R        group of formula I.

The invention provides for a method of preparing an LNA oligonucleotidewhich is essentially free from a +28 adduct comprising the steps of:

a) Incorporating at least one LNA-G monomer comprising an acyl protectedexocyclic nitrogen into an oligonucleotide;

b) Incorporating at least one optionally protected aliphatic amine groupinto the oligonucleotide;

c) deprotecting the acyl protected exocyclic nitrogen of the at leastone LNA-G monomer by removal of the acyl protection group;

-   -   wherein steps a) and b) can occur in either order.

The invention provides for a method of preparing an LNA oligonucleotidecomprising the steps of:

a) Incorporating at least one G monomer comprising an acyl protectedexocyclic nitrogen into an oligonucleotide;

b) Incorporating at least one optionally protected aliphatic amine groupinto the oligonucleotide;

c) deprotecting the acyl protected exocyclic nitrogen of the at leastone G monomer by removal of the acyl protection group;

-   -   wherein steps a) and b) can occur in either order.

The invention provides for a LNA oligonucleotide which comprises atleast one LNA-G monomer comprising an acyl protected exocyclic nitrogenand at least one optionally protected aliphatic amine group, whereinsaid LNA oligonucleotide is attached to a solid support.

The invention provides for a LNA oligonucleotide which comprises atleast one G monomer comprising an acyl protected exocyclic nitrogen andat least one optionally protected aliphatic amine group, wherein saidLNA oligonucleotide is attached to a solid support.

The invention provides for an LNA oligonucleotide which comprises atleast one LNA-G monomer and at least one optionally protected aliphaticamine group, wherein said LNA oligonucleotide, wherein saidoligonucleotide is essentially free of +28 adduct.

The invention provides for an LNA oligonucleotide which comprises atleast one G monomer and at least one optionally protected aliphaticamine group, wherein said LNA oligonucleotide, wherein saidoligonucleotide is essentially free of +28 adduct.

The invention provides for a pharmaceutical composition comprising anLNA oligomer conjugate which comprises an LNA-G monomer and an aliphaticamine linker positioned between the 5′ nucleotide of LNA oligomer and aconjugate moiety, and a pharmaceutically acceptable diluent, carrier oradjuvant, wherein said composition is essentially free of +28 adduct.

The invention provides for a pharmaceutical composition comprising anLNA oligomer conjugate which comprises an G monomer and an aliphaticamine linker positioned between the 5′ nucleotide of LNA oligomer and aconjugate moiety, and a pharmaceutically acceptable diluent, carrier oradjuvant, wherein said composition is essentially free of +28 adduct.

The invention provides for the use of an LNA-G monomer comprising anacyl protected exocyclic nitrogen for use in the synthesis of analiphatic amine containing LNA oligonucleotide.

The invention provides for the use of an LNA-G monomer comprising anacyl protected exocyclic nitrogen for use in the synthesis of analiphatic amine containing LNA oligonucleotide conjugate.

In some embodiments the LNA-G monomer is a monomer of formula I:

wherein R may be selected from an optionally substituted alkyl-,alkenyl-, alkynyl-, cycloalkyl- or aryl-group, preferably from anoptionally substituted C₁₋₆-alkyl-, C₂₋₆-alkenyl-, C₂₋₆-alkinyl-,C₃₋₇-cycloalkyl- or phenyl-group.

If substituted, the R group may be mono or poly substituted, for examplewith one or more substituents selected from the group consisting ofhalogen, C₁₋₆-alkyl, C₂₋₆-alkenyl, C₂₋₆-alkynyl, C₁₋₆-alkoxy, optionallysubstituted aryloxy or optionally substituted aryl. Aryl includes phenyland the optional substituents for aryl are as above.

FIGURES

FIG. 1: Examples of commercially available amino-linkers comprising analiphatic amine group.

FIG. 2: Examples of GalNAc conjugates.

FIG. 3: Mass spectra of FL and FL+28 (five-fold charged ion displayed).

FIG. 4: MS/MS (MS2MS3 fragmentation pattern (excerpt mass range 500-1500Da) of b4 ions. Peaks with mass difference +28 Da and correspondingpeaks in the unmodified molecule are indicated.

FIG. 5: Structure of the b4-fragment and the resulting a2-Base (a2-B)and the b4-Cytosine (b4-C) fragment from MS3 experiment.

DETAILED DESCRIPTION

The invention provides a method of preparing an oligonucleotide whichcomprises at least one LNA-G nucleoside and an aliphatic amine group. Ithas been found by the present inventors that a +28 Da adduct (referredto as the +28 adduct herein) is formed when deprotecting DMF protectedLNA-G in the presence of an aliphatic amine. The formation of the +28adduct may be avoided by using an acyl protection group. The presence of+28 adduct may be identified using mass spectroscopy, by a contaminantin the oligonucleotide with +28 molecular weight. As is illustrated inthe example the +28 adduct may be measured using mass spectroscopy, andmay have a MW of about +28, for example the +28 adduct may have a MW of+27.5-+28.5 (Da), such as +27.9-+28.1 (Da). This impurity is difficultto separate from the desired oligonucleotide product, and requiresadditional down-stream purification steps, increasing the cost ofproduction and dramatically reducing the oligonucleotide yield. It istherefore highly desirable to avoid the production of the +28 adduct,and it has been found by the present inventors that this may be achievedby using G-protection groups other than DMF, in particular an acyl orcarbamate protection group.

An LNA oligonucleotide is an oligonucleotide which comprises at leastone LNA nucleoside. The invention therefore relates to methods ofpreparing LNA antisense oligonucleotides which comprise at least oneLNA-G monomer and at least one aliphatic amine group. The LNAoligonucleotide may be an antisense oligonucleotide. The termoligonucleotide as used herein is defined as it is generally understoodby the skilled person as a molecule comprising two or more covalentlylinked nucleosides. For use as an antisense oligonucleotide,oligonucleotides are typically synthesised as 7-30 nucleotides inlength.

The term “antisense oligonucleotide” as used herein is refers tooligonucleotides capable of modulating expression of a target gene byhybridizing to a target nucleic acid, in particular to a contiguoussequence on a target nucleic acid. An antisense oligonucleotide can alsobe defined by it's complementary to a target nucleic acid. Antisenseoligonucleotides are single stranded. Antisense oligonucleotides are notessentially double stranded and are not therefore siRNAs. An antisenseoligonucleotide comprises a contiguous nucleotide which is complementaryto a target nucleic acid. Antisense oligonucleotides typically compriseone or more modified internucleoside linkages, and may by way of anon-limiting example be in the form of a a LNA gapmer or a mixed winggapmer. In other embodiments the oligonucleotide may be an LNA mixmers(LNA and non-LNA nucleotides, e.g. LNA and DNA (see e.g. WO2007/112754hereby incorporated by reference), or LNA and 2′-O-MOE nucleotides, orLNA, DNA and 2′O-MOE nucleotides), or a LNA totalmers (only LNAnucleotides—see. E.g. WO2009/043353 hereby incorporated by reference).

The term “modified internucleoside linkage” is defined as generallyunderstood by the skilled person as linkages other than phosphodiester(PO) linkages, that covalently couples two nucleosides together.Modified internucleoside linkages are particularly useful in stabilizingoligonucleotides for in vivo use, and may serve to protect againstnuclease cleavage. A phosphorothioate internucleoside linkage isparticularly useful due to nuclease resistance, beneficialpharmakokinetics and ease of manufacture. In some embodiments at least70%, such as at least 80 or such as at least 90% of the internucleosidelinkages in the oligonucleotide, or contiguous nucleotide sequencethereof, are phosphorothioate. In some embodiments all of theinternucleoside linkages of the oligonucleotide, or contiguousnucleotide sequence thereof, are phosphorothioate. Furtherinternucleoside linkers are disclosed in WO2009/124238 (incorporatedherein by reference).

The term “essentially free” is defined by the level of +28 adduct isless than 5%, such as less than 1%, such as less than 0.5%, such as lessthan 0.1%, of the oligonucleotide composition prepared by the methods ofthe invention. An oligonucleotide which is “essentially free” of the +28adduct may therefore comprise a small amount of +28 adduct, and in someembodiments the level of +28 adduct may be below the level of detectionusing mass spectroscopy. In term essentially free comprises theembodiment where the oligonucleotide product is free of +28 adduct.

The term nucleobase includes the purine (e.g. adenine and guanine) andpyrimidine (e.g. uracil, thymine and cytosine) moiety present innucleosides and nucleotides which form hydrogen bonds in nucleic acidhybridization. In the context of the present invention the termnucleobase also encompasses modified nucleobases which may differ fromnaturally occurring nucleobases, but are functional during nucleic acidhybridization. In some embodiments the nucleobase moiety is modified bymodifying or replacing the nucleobase. In this context “nucleobase”refers to both naturally occurring nucleobases such as adenine, guanine,cytosine, thymidine, uracil, xanthine and hypoxanthine, as well asnon-naturally occurring variants. Such variants are for exampledescribed in Hirao et al (2012) Accounts of Chemical Research vol 45page 2055 and Bergstrom (2009) Current Protocols in Nucleic AcidChemistry Suppl. 37 1.4.1.

Nucleotides are the building blocks of oligonucleotides andpolynucleotides, and for the purposes of the present invention includeboth naturally occurring and non-naturally occurring nucleotides. Innature, nucleotides, such as DNA and RNA nucleotides comprise a ribosesugar moiety, a nucleobase moiety and one or more phosphate groups(which is absent in nucleosides). Modified nucleosides and nucleotidesare modified as compared to the equivalent DNA or RNA nucleoside/tide bythe introduction of a modification to the ribose sugar moiety, thenucleobase moiety, or in the case of modified nucleotides, theinternucleoside linkage. Nucleosides and nucleotides may alsointerchangeably be referred to as “units” or “monomers”.

The term “modified nucleoside” or “nucleoside modification” as usedherein refers to nucleosides modified as compared to the equivalent DNAor RNA nucleoside by the introduction of one or more modifications ofthe sugar moiety or the (nucleo)base moiety. The term modifiednucleoside may also be used herein interchangeably with the term“nucleoside analogue” or modified “units” or modified “monomers”.Examples of modified nucleosides are described in the separate section“Oligomer modifications” and its sub-sections.

Acyl Protected Exocyclic Nitrogen

The exocyclic nitrogen group of guanine is illustrated below(encircled). This group is protected by an acyl group during steps a)and b) of the method of the invention, and is removed during thedeprotection step c).

The Aliphatic Amine Group

An aliphatic amine is an amine where there are no aromatic ringsdirectly on the nitrogen atom, and is therefore typically a nonnucleosidic amine group. A nucleosidic amine group is an amine groupwhere the nitrogen atom of the amine is directly bound to the aromaticring of a purine or pyrimidine base. The aliphatic amine group may be aprimary amine or a secondary amine. In some embodiments, the aliphaticamine group is selected from the group consisting of an amino alkyl,alkylamino alkyl, piperidine, piperazine, pyrrolidine, and imidazole. Insome embodiments, the aliphatic amine group is selected from the groupconsisting of 5′-TFA-Amino-Modifier-C5-CE Phosphoramidite,5′-TFA-Amino-Modifier C6-CE Phosphoramidite,11-(trifluoroacetamido)-3,6,9-trioxaundecan-1-yl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite,5′-TFA-Amino-Modifier-C12-CE Phosphoramidite, Amino-Modifier C2-dT-CEPhosphoramidite, Amino-Modifier C6-dA-CE Phosphoramidite, Amino-ModifierC6-dA-CE Phosphoramidite, Amino-Modifier C6-dT-CE Phosphoramidite,N2-Amino-Modifier C6 dG, Fmoc Amino-Modifier C6 dT, 3′-Amino-Modifier C7CPG 1000, 3′-Amino-Modifier C6-dC CPG, 3′-Amino-Modifier C6-dC CPG,3′-PT-Amino-Modifier C6 CPG, 3′-Amino-Modifier C6-dT CPG, PC5′-Amino-Modifier-CE Phosphoramidite, 5′-Amino-Modifier C6-PDA,5′-Amino-Modifier C12-PDA, 5′-Amino-Modifier TEG PDA, Amino-ModifierSerinol, 3′-Amino-Modifier Serinol CPG

In some embodiments, the aliphatic amine group is in the form of anamino linker (i.e. the amino linker comprises the aliphatic aminegroup). Examples of commercially available amino linkers are shown inFIG. 1. In some embodiments, the amino linker is an aminoalkyl linker,such as a C₂₋₁₂ aminoalkyl linker, for example an amino hexyl linker.

In some embodiments the aliphatic amino group is protected, for examplewith a protection group selected from the list comprising oftrifluoroacetyl (TFA), trichloroacetyl (TCA), monomethoxytrityl (MMT),dimethoxytrityl (DMT), fluorenylmethyloxycarbonyl (Fmoc), phtalimide or2-(methylaminocarbonyl)-benzoate. In some embodiments the amineprotection group, when present, is removed prior to or during thedeprotection step c).

It is recognised that some aliphatic amine protection groups may survivestep c), and as such they provide an alternative method of avoiding the+28 adduct. The invention therefore provides an alternative method ofpreparing a LNA oligonucleotide comprising the steps of:

-   -   a) Incorporating at least one protected exocyclic nitrogen LNA-G        monomer into an oligonucleotide    -   b) Incorporating at least one protected aliphatic amine group        into the oligonucleotide    -   c) deprotecting the at least one exocyclic nitrogen LNA-G        monomer containing oligonucleotide by removal of the exocyclic        nitrogen protection group    -   d) subsequent to step c), deprotecting the aliphatic amine        group.

The deprotection step c) may comprise exposure of the oligonucleotide toammonium hydroxide, and suitably the aliphatic amine protection groupare not cleaved under the deprotection conditions of step c) (i.e. byammonium hydroxide treatment). In the above method the exocyclicnitrogen protection group may be an acyl group or may be anotherprotection group such as dimethylformamide (DMF). The aliphatic amineprotection group may, for example, be selected from the group consistingof TFA, monomethoxytrityl (MMT), DMT, Fmoc, phtalimide or2-(methylaminocarbonyl)-benzoate

In some embodiments, the aliphatic amine group, such as the aminolinker, is attached to a solid support used for oligonucleotidesynthesis. The cleavage of the oligonucleotide from the solid support(which may be during step c)) will therefore result in the cleavage ofthe alpiphatic amine group from the solid support and thereby release ofthe oligonucleotide from the solid support. In some embodiments, thealiphatic amine group is incorporated into the oligonucleotide via theincorporation of an amino-modified monomer. In some embodiments, thealiphatic amino-modified monomer is a phosphoramidite, a H-phosphonateor a phosphotriester monomer. In some embodiments, the amino-modifiedmonomer is a phosphoramidite. Examples of such amino-modified monomersare shown in FIG. 1.

The aliphatic amine group may be incorporated into the oligonucelotidevia any suitable oligonucleotide synthesis method, such as H-phosphonatesynthesis, phosphodiester synthesis, phosphotriester synthesis,phosphite trimester synthesis or phosphoramidite oligonucleotidesynthesis. In some embodiments the aliphatic amine group is incorporatedinto the oligonucleotide as phosphoramidite, a H-phosphonate or aphosphotriester. In some embodiments the aliphatic amine group isincorporated into the oligonucleotide during phosphoramiditeoligonucleotide synthesis.

The Acyl Protection Group

The use of an acyl protection group on the exocyclic nitrogen of Gresidues allows for the avoidance of the +28 adduct in methods ofsynthesis of aliphatic amine containing oligonucleotides.

Some non-limiting examples of suitable acyl protection groups on theexocyclic nitrogen of the LNA-G monomer(s) may be selected from thegroup consisting of Isobuturyl (iBu), Acetyl (Ac), Phenoxyacetyl (PAC),p-Isopropylphenoxyacetyl (iPrPAC), phenylacetyl, Isopropyloxyacetyl,methoxyacetyl, benzoyl, p-methoxyphenylacetyl, diphenylacetyl,cyclohexylcarbonyl, 1,1-dimethylpropanoyl, andp-tert-Butyl-phenoxyacetyl.

In some embodiments, the acyl protection group on the exocyclic nitrogenof the LNA-G monomer(s) is selected from the group consisting ofIsobuturyl (iBu), Acetyl (Ac), Phenoxyacetyl (PAC), &p-Isopropylphenoxyacetyl (iPrPAC).

In an alternative embodiment, the acyl protection group may be replacedwith a carbamate protection group.

The LNA G Monomer

The term LNA-G refers to a nucleoside which comprises a 2′-4′ biradicalin the furanose ring and a guanine nucleobase. The LNA-G monomer may beincorporated into the oligonucleotide via any suitable oligonucleotidesynthesis method, such as H-phosphonate synthesis, phosphodiestersynthesis, phosphotriester synthesis, phosphite trimester synthesis orphosphoramidite oligonucleotide synthesis. In some embodiments the LNA-Gmonomer(s) is incorporated into the oligonucleotide as phosphoramidite,a H-phosphonate or a phosphotriester. In some embodiments the LNA-Gmonomer is incorporated into the oligonucleotide during phosphoramiditeoligonucleotide synthesis.

In some embodiments, the LNA oligonucleotide comprises at least 1 Gmonomers, such as at least 2 G monomers, such as at least 3 G monomers,such as at least 4 G monomers.

Oligonucleotide Synthesis (Step a or Step a) and b))

The method of preparing an oligonucleotide may utilise any suitableoligonucleotide synthesis method, such as H-phosphonate synthesis,phosphodiester synthesis, phosphotriester synthesis, phosphite trimestersynthesis or phosphoramidite oligonucleotide synthesis. The LNA-Gmonomer and optionally the aliphatic amine group (optionally protected)may in a form which allows for incorporation into the oligonucleotideduring such standard oligonucleotide methods (e.g. as aphosphoramidite).

Deprotection Step c)

Step c) of the method of the invention comprises the removal of the acylprotection group from the exocyclic nitrogen in the LNA-G monomerincorporated into the oligonucleotide. Deprotection may further comprisethe removal of other base protection groups, and optionally the removalof the aliphatic amine protection group, when present. During solidphase synthesis of oligonucleotides, the deprotection step may furtherresult in the cleavage of the oligonucleotide from the solid support.deprotection (and optionally cleavage) of the oligonucleotide mayperformed in the presence of ammonia, such as using a solutioncomprising ammonium hydroxide. For example, concentrated ammoniumhydroxide may be used (e.g. (28 to 33% NH3 in water), or a 1:1 mixtureof ammonium hydroxide and aqueous methylamine (AMA). Other deprotectionmethods are known in the art.

Conjugation Step

The oligonucleotides synthesised according to the method of theinvention are particularly useful for making oligonucleotide conjugatesas the aliphatic amine group provides an amenable conjugation site.Oligonucleotide conjugates comprise an oligonucleotide which iscovalently linked to a non-nucleoside moiety, which may for example be alipid, a sterol, a carbohydrate, a peptide and a protein. Examples ofconjugate moieties are disclosed in WO2014/076195 and Wo2014/179620,which are hereby incorporated by reference.

In some embodiments, the method of the invention comprises an additionalstep performed subsequent to step c) which comprises comprisesincorporating a conjugate group onto the aliphatic primary amine group.

The invention therefore provides for a method of preparing a LNAoligonucleotide conjugate comprising the steps of:

a) Incorporating at least one nitrogen LNA-G monomer comprising an acylprotected exocyclic nitrogen into an oligonucleotide

b) Incorporating at least one optionally protected aliphatic amine groupinto the oligonucleotide

c) deprotecting the at least one acyl protected exocyclic nitrogen ofthe at least one LNA-G monomer oligonucleotide by removal of the acylprotection group.

d) Incorporating a conjugate group onto the aliphatic amine group.wherein steps a) and b) can occur in either order or simultaneously.

In some embodiments the conjugate moiety is a carbohydrate, such as aN-Acetylgalactosamine(GalNAc) conjugates, see WO2014/118267, which ishereby incorporated by reference. GalNaC conjugates are useful inenhancing uptake into cells, such as liver cells, and are typically useas a GalNAc cluster, such as a trivalent GalNAc cluster. Examples ofGalNAc conjugates which may be incorporated into oligonucleotides usingthe methods of the invention are illustrated in FIG. 2.

Linkers

A linkage or linker is a connection between two atoms that links onechemical group or segment of interest to another chemical group orsegment of interest via one or more covalent bonds. Conjugate moietiescan be attached to the oligonucleotide directly or through a linkingmoiety (e.g. linker or tether). Linkers serve to covalently connect athird region, e.g. a conjugate moiety to a first region, e.g. anoligonucleotide (region A). In the context of the present invention thelinker may comprise the aliphatic amine group, such as a primary orsecondary aliphatic amine group. In some embodiments the linker is analiphatic amino alkyl, such as a C₂-C₃₆ aliphatic amino alkyl group,including, for example C₆ to C₁₂ aliphatic amino alkyl groups. In someembodiments the linker is a C₆ aliphatic amino alkyl group. In someembodiments the oligonucleotide comprises a region of DNA phosphodiesternucleotides, e.g. 1-5 DNA PO nucleotides which are positioned betweenthe antisense oligonucleotide and the aliphatic amino linker—seeWO2014/076195 hereby incorporated by reference.

Locked Nucleic Acid Nucleosides (LNA).

LNA nucleosides are modified nucleosides which comprise a linker group(referred to as a biradicle or a bridge) between C2′ and C4′ of theribose sugar ring of a nucleotide. These nucleosides are also termedbridged nucleic acid or bicyclic nucleic acid (BNA) in the literature.

In some embodiments, the modified nucleoside or the LNA nucleosides ofthe oligomer of the invention has a general structure of the formula Ior II:

wherein W is selected from —O—, —S—, —N(R^(a))—, —C(R^(a)R^(b))—, suchas, in some embodiments —O—;

B designates a nucleobase or modified nucleobase moiety;

Z designates an internucleoside linkage to an adjacent nucleoside, or a5′-terminal group;

Z designates an internucleoside linkage to an adjacent nucleoside, or a3′-terminal group;

X designates a group selected from the list consisting of—C(R^(a)R^(b))—, —C(R^(a))═C(R^(b))—, —C(R^(a))═N—, —O—, —Si(R^(a))₂—,—S—, —SO₂—, —N(R^(a))—, and >C═Z

-   -   In some embodiments, X is selected from the group consisting of:        —O—, —S—, NH—, NR^(a)R^(b), —CH₂—, CR^(a)R^(b), —C(═CH₂)—, and        —C(═CR^(a)R^(b))—    -   In some embodiments, X is —O—

Y designates a group selected from the group consisting of—C(R^(a)R^(b))—, —C(R^(a))═C(R^(b))—, —C(R^(a))═N—, —O—, —Si(R^(a))₂—,—S—, —SO₂—, —N(R^(a))—, and >C═Z

-   -   In some embodiments, Y is selected from the group consisting of:        —CH₂—, —C(R^(a)R^(b))—, —CH₂CH₂—, —C(R^(a)R^(b))—C(R^(a)R^(b))—,        —CH₂CH₂CH₂—, —C(R^(a)R^(b))C(R^(a)R^(b))C(R^(a)R^(b))—,        —C(R^(a))═C(R^(b))—, and —C(R^(a))═N—    -   In some embodiments, Y is selected from the group consisting of:        —CH₂—, —CHR^(a)—, —CHCH₃—, CR^(a)R^(b)—

or -X-Y- together designate a bivalent linker group (also referred to asa radicle) together designate a bivalent linker group consisting of 1,2, or 3 groups/atoms selected from the group consisting of—C(R^(a)R^(b))—, —C(R^(a))═C(R^(b))—, —C(R^(a))═N—, —O—, —Si(R^(a))₂—,—S—, —SO₂—, —N(R^(a))—, and >C═Z,

-   -   In some embodiments, -X-Y- designates a biradicle selected from        the groups consisting of: —X—CH₂—, —X—CR^(a)R^(b)—, —X—CHR^(a−),        —X—C(HCH₃)⁻, —O—Y—, —O—CH₂—, —S—CH₂—, —NH—CH₂—, —O—CHCH₃—,        —CH₂—O—CH₂, —O—CH(CH₃CH₃)—, —O—CH₂—CH₂—, OCH₂—CH₂—CH₂—,        —O—CH₂OCH₂—, —O—NCH₂—, —C(═CH₂)—CH₂—, —NR^(a)—CH₂—, N—O—CH₂,        —S—CR^(a)R^(b)— and —S—CHR^(a)—.    -   In some embodiments -X-Y- designates —O—CH₂— or —O—CH(CH₃)—.

wherein Z is selected from —O—, —S—, and —N(R^(a))—,

and R^(a) and, when present R^(b), each is independently selected fromhydrogen, optionally substituted C₁₋₆-alkyl, optionally substitutedC₂₋₆-alkenyl, optionally substituted C₂₋₆-alkynyl, hydroxy, optionallysubstituted C₁₋₆-alkoxy, C₂₋₆-alkoxyalkyl, C₂₋₆-alkenyloxy, carboxy,C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl,aryloxy, arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl,heteroaryloxy, heteroarylcarbonyl, amino, mono- and di(C₁₋₆-alkyl)amino,carbamoyl, mono- and di(C₁₋₆-alkyl)-amino-carbonyl,amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, where aryl and heteroaryl maybe optionally substituted and where two geminal substituents R^(a) andR^(b) together may designate optionally substituted methylene (═CH₂),wherein for all chiral centers, asymmetric groups may be found in eitherR or S orientation.

wherein R¹, R², R³, R⁵ and R^(5*) are independently selected from thegroup consisting of: hydrogen, optionally substituted C₁₋₆-alkyl,optionally substituted C₂₋₆-alkenyl, optionally substitutedC₂₋₆-alkynyl, hydroxy, C₁₋₆-alkoxy, C₂₋₆-alkoxyalkyl, C₂₋₆-alkenyloxy,carboxy, C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl, formyl, aryl,aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, where aryl and heteroaryl maybe optionally substituted, and where two geminal substituents togethermay designate oxo, thioxo, imino, or optionally substituted methylene.

-   -   In some embodiments R¹, R², R³, R⁵ and R^(5*) are independently        selected from C₁₋₆alkyl, such as methyl, and hydrogen.    -   In some embodiments R¹, R², R³, R⁵ and R^(5*) are all hydrogen.    -   In some embodiments R¹, R², R³, are all hydrogen, and either R⁵        and R^(5*) is also hydrogen and the other of R⁵ and R^(5*)is        other than hydrogen, such as C₁₋₆ alkyl such as methyl.    -   In some embodiments, R^(a) is either hydrogen or methyl. In some        embodiments, when present, R^(b) is either hydrogen or methyl.    -   In some embodiments, one or both of R^(a) and R^(b) is hydrogen    -   In some embodiments, one of R^(a) and R^(b) is hydrogen and the        other is other than hydrogen    -   In some embodiments, one of R^(a) and R^(b) is methyl and the        other is hydrogen    -   In some embodiments, both of R^(a) and R^(b) are methyl.

In some embodiments, the biradicle -X-Y- is —O—CH₂—, W is O, and all ofR¹, R², R³, R⁵ and R^(5*) are all hydrogen. Such LNA nucleosides aredisclosed in WO99/014226, WO00/66604, WO98/039352 and WO2004/046160which are all hereby incorporated by reference, and include what arecommonly known as beta-D-oxy LNA and alpha-L-oxy LNA nucleosides.

In some embodiments, the biradicle -X-Y- is —S—CH₂—, W is O, and all ofR¹, R², R³, R⁵ and R^(5*) are all hydrogen. Such thio LNA nucleosidesare disclosed in WO99/014226 and WO2004/046160 which are herebyincorporated by reference.

In some embodiments, the biradicle -X-Y- is —NH—CH₂—, W is O, and all ofR¹, R², R³, R⁵ and R^(5*) are all hydrogen. Such amino LNA nucleosidesare disclosed in WO99/014226 and WO2004/046160 which are herebyincorporated by reference. In some embodiments, the biradicle -X-Y- is—O—CH₂—CH₂— or —O—CH₂—CH₂— CH₂—, W is O, and all of R¹, R², R³, R⁵ andR^(5*) are all hydrogen. Such LNA nucleosides are disclosed inWO00/047599 and Morita et al, Bioorganic & Med.Chem. Lett. 12 73-76,which are hereby incorporated by reference, and include what arecommonly known as 2′-O-4′O-ethylene bridged nucleic acids (ENA).

In some embodiments, the biradicle -X-Y- is —O—CH₂—, W is O, and all ofR¹, R², R³, and one of R⁵ and R^(5*) are hydrogen, and the other of R⁵and R^(5*) is other than hydrogen such as C₁₋₆ alkyl, such as methyl.Such 5′ substituted LNA nucleosides are disclosed in WO2007/134181 whichis hereby incorporated by reference.

In some embodiments, the biradicle -X-Y- is —O—CR^(a)R^(b)—, wherein oneor both of R^(a) and R^(b) are other than hydrogen, such as methyl, W isO, and all of R¹, R², R³, and one of R⁵ and R^(5*) are hydrogen, and theother of R⁵ and R^(5*) is other than hydrogen such as O₁₋₆ alkyl, suchas methyl. Such bis modified LNA nucleosides are disclosed inWO2010/077578 which is hereby incorporated by reference.

In some embodiments, the biradicle -X-Y- designate the bivalent linkergroup —O—CH(CH₂OCH₃)— (2′ O-methoxyethyl bicyclic nucleic acid—Seth atal., 2010, J. Org. Chem., 2010, 75 (5), pp 1569-1581). In someembodiments, the biradicle -X-Y-designate the bivalent linker group—O—CH(CH₂CH₃)— (2′O-ethyl bicyclic nucleic acid —Seth at al., 2010, J.Org. Chem). In some embodiments, the biradicle -X-Y- is —O—CHR^(a)—, Wis O, and all of R¹, R², R³, R⁵ and R^(5*) are all hydrogen. Such 6′substituted LNA nucleosides are disclosed in WO10036698 and WO07090071which are both hereby incorporated by reference.

In some embodiments, the biradicle -X-Y- is —O—CH(CH₂OCH₃)—, W is O, andall of R¹, R², R³, R⁵ and R^(5*) are all hydrogen. Such LNA nucleosidesare also known as cyclic MOEs in the art (cMOE) and are disclosed inWO07090071.

In some embodiments, the biradicle -X-Y- designate the bivalent linkergroup —O—CH(CH₃)—.—in either the R- or S-configuration. In someembodiments, the biradicle -X-Y-together designate the bivalent linkergroup —O—CH₂—O—CH₂— (Seth at al., 2010, J. Org. Chem). In someembodiments, the biradicle -X-Y- is —O—CH(CH₃)—, W is O, and all of R¹,R², R³, R⁵ and R^(5*) are all hydrogen. Such 6′ methyl LNA nucleosidesare also known as cET nucleosides in the art, and may be either (S)cETor (R)cET stereoisomers, as disclosed in WO07090071 (beta-D) andWO2010/036698 (alpha-L) which are both hereby incorporated byreference).

In some embodiments, the biradicle -X-Y- is —O—CR^(a)R^(b)—, wherein inneither R^(a) or R^(b) is hydrogen, W is O, and all of R¹, R², R³, R⁵and R^(5*) are all hydrogen. In some embodiments, R^(a) and R^(b) areboth methyl. Such 6′ di-substituted LNA nucleosides are disclosed in WO2009006478 which is hereby incorporated by reference.

In some embodiments, the biradicle -X-Y- is —S—CHR^(a)—, W is O, and allof R¹, R², R³, R⁵ and R^(5*) are all hydrogen. Such 6′ substituted thioLNA nucleosides are disclosed in WO11156202 which is hereby incorporatedby reference. In some 6′ substituted thio LNA embodiments R^(a) ismethyl.

In some embodiments, the biradicle -X-Y- is —C(═CH2)-C(R^(a)R^(b))—,such as —C(═CH₂)—CH₂—, or —C(═CH₂)—CH(CH₃)—W is O, and all of R¹, R²,R³, R⁵ and R^(5*) are all hydrogen. Such vinyl carbo LNA nucleosides aredisclosed in WO08154401 and WO09067647 which are both herebyincorporated by reference.

In some embodiments the biradicle -X-Y- is —N(—OR^(a))—, W is O, and allof R¹, R², R³, R⁵ and R^(5*) are all hydrogen. In some embodiments R^(a)is C₁₋₆ alkyl such as methyl. Such LNA nucleosides are also known as Nsubstituted LNAs and are disclosed in WO2008/150729 which is herebyincorporated by reference. In some embodiments, the biradicle -X-Y-together designate the bivalent linker group —O—NR^(a)—CH₃— (Seth atal., 2010, J. Org. Chem). In some embodiments the biradicle -X-Y- is—N(R^(a))—, W is O, and all of R¹, R², R³, R⁵ and R^(5*) are allhydrogen. In some embodiments R^(a) is C₁₋₆ alkyl such as methyl.

In some embodiments, one or both of R⁵ and R^(5*) is hydrogen and, whensubstituted the other of R⁵ and R^(5*) is C₁₋₆ alkyl such as methyl. Insuch an embodiment, R¹, R², R³, may all be hydrogen, and the biradicle-X-Y- may be selected from —O-CH2- or —O—C(HCR^(a))—, such as—O—C(HCH3)-.

In some embodiments, the biradicle is —CR^(a)R^(b)—O—CR^(a)R^(b)—, suchas CH₂—O—CH₂—, W is O and all of R¹, R², R³, R⁵ and R^(5*) are allhydrogen. In some embodiments R^(a) is C₁₋₆ alkyl such as methyl. SuchLNA nucleosides are also known as conformationally restrictednucleotides (CRNs) and are disclosed in WO2013036868 which is herebyincorporated by reference.

In some embodiments, the biradicle is —O—CR^(a)R^(b)—O—CR^(a)R^(b)—,such asO—CH₂—O—CH₂—, W is O and all of R¹, R², R³, R⁵ and R^(5*) are allhydrogen. In some embodiments R^(a) is C₁₋₆ alkyl such as methyl. SuchLNA nucleosides are also known as COC nucleotides and are disclosed inMitsuoka et al., Nucleic Acids Research 2009 37(4), 1225-1238, which ishereby incorporated by reference.

It will be recognized than, unless specified, the LNA nucleosides may bein the beta-D or alpha-L stereoisoform.

Certain examples of LNA nucleosides are presented in Scheme 1.

As illustrated in the examples, in some embodiments of the invention theLNA nucleosides in the oligonucleotides are or comprise beta-D-oxy-LNAnucleosides.

Gapmer

The term gapmer as used herein refers to an antisense oligonucleotidewhich comprises a region of RNase H recruiting oligonucleotides (gap)which is flanked 5′ and 3′ by one or more affinity enhancing modifiednucleosides (flanks). Various gapmer designs are described herein.Headmers and tailmers are oligonucleotides capable of recruiting RNase Hwhere one of the flanks are missing, i.e. only one of the ends of theoligonucleotide comprises affinity enhancing modified nucleosides. Forheadmers the 3′ flank is missing (i.e. the 5′ flanc comprise affinityenhancing modified nucleosides) and for tailmers the 5′ flank is missing(i.e. the 3′ flank comprises affinity enhancing modified nucleosides).

LNA Gapmer

The term LNA gapmer is a gapmer oligonucleotide wherein at least one ofthe affinity enhancing modified nucleosides is an LNA nucleoside.

Mixed Wing Gapmer

The term mixed wing gapmer refers to a LNA gapmer wherein the flankregions comprise at least one LNA nucleoside and at least one non-LNAmodified nucleoside, such as at least one 2′ substituted modifiednucleoside, such as, for example, 2′-O-alkyl-RNA, 2′-O-methyl-RNA,2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA (MOE), 2′-amino-DNA, 2′-Fluoro-DNA,arabino nucleic acid (ANA), 2′-fluoro-ANA and 2′-F-ANA nucleoside(s). Insome embodiments the mixed wing gapmer has one flank which comprises LNAnucleosides (e.g. 5′ or 3′) and the other flank (3′ or 5′ respectfully)comprises 2′ substituted modified nucleoside(s).

Length

When referring to the length of a nucleotide molecule as referred toherein, the length corresponds to the number of monomer units, i.e.nucleotides, irrespective as to whether those monomer units arenucleotides or nucleotide analogues. With respect to nucleotides, theterms monomer and unit are used interchangeably herein.

The process of the present invention is particularly suitable for thepurification of short oligonucleotides, for example, consisting of 7 to30 nucleotides, such as 7-10, such as 7, 8, 9, 10 or 10 to 20nucleotides, such as 12 to 18 nucleotides, for example, 12, 13, 14, 15,16, 17 or 18 nucleotides.

EXAMPLES Example 1

Synthesis of isobuturyl protected LNA-G phosporamidite is described inKoshkin et al, Tetrahedron (1998), 54(14), 3607-3630.

Crude phosphorthioate oligonucleotides were synthesized in DMT-OFF modeat 20 μmol scale on a NittoPhase UnyLinker 200 polystyrene support bystandard phosphoramidite chemistry, except for oligonucleotides in entry17-18 which were synthesized on a 3′-Amino-Modifier C7 CPG support.4,5-dicyanoimidazole was used as activator and xanthane hydride was usedfor thiooxidation. Standard DNA phosphoramidites with benzoyl protectedA and C were used. LNA phosphoramidites with benzoyl protected A and5-methyl-C were used. LNA-G was DMF- or iBu-protected as indicated inthe table below.

5′-TFA-Amino-Modifier C6-CE phosphoramidite, available from LinkTechnologies, Lanakshire, Scotland, was used to introduce the6-aminohexyl linker (AM-C6) in the 5′-end.

Amino-Modifier C6-dT-CE Phosphoramidite(5′-Dimethoxytrityl-5-[N-(trifluoroacetylaminohexyl)-3-acrylimido]-2′-deoxyUridine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite),available from Glen Research, Sterling, Va., was used to introduce the5-[N-(aminohexyl)-3-acrylimido]-2′-deoxyuridine linker (t^(AMC6)).

11-(trifluoroacetamido)-3,6,9-trioxaundecan-1-yl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, available from Link Technologies,Lanakshire, Scotland, was used to introduce the11-amino-3,6,9-trioxaundecan-1-yl group in the 5′-end (AM-TEG).

3′-Amino-Modifier C7 CPG(2-Dimethoxytrityloxymethyl-6-fluorenylmethoxycarbonylamino-hexane-1-succinoyl)-longchain alkylamino-CPG), available from Glen Research, Sterling, Va., wasused to introduce the 6-amino-2-(hydroxymethyl)-hexyl group (AM-C7) inthe 3′-end. In this case, after end of synthesis the support was treatedwith first a solution of diethylamine and then with 20% piperidine inDMF to remove the Fmoc group.

After end synthesis, the support was washed with a solution ofdiethylamine and then suspended in 1 mL concentrated ammonium hydroxideat 60° C. over night. The support was filtered off and the solution wasevaporated to dryness under vacuum. The crude material was analyzed byUPLC-MS and ratios of +28 Da impurity compared to correct product wasestimated by peak area at 260 nm.

Entry Amount of (SEQ +28 Da ID NO) Sequence LNA-G impurity 15′-AM-C6-TGctatttcatctTGG-3′ DMF 10.0% 2 5′-AM-C6-TTctatttcatctTCT-3′ 0.0% 3 5′-AM-C6-AtGcTcGaTG-3′ DMF  9.3% 4 5′-AM-C6-AtGcTcGaTG-3′ iBu 0.0% 5 5′-AM-C6-GCTGATGAGT-3′ DMF  7.5% 6 5′-AM-C6-GCTGATGAGT-3′ iBu 0.0% 7 5′-AM-C6-CGGtaacttcaGCA-3′ DMF 15.3% 85′-AM-C6-CGGtaacttcaGCA-3′ iBu  0.0% 9 5′-AM-C6-atGTtcGGcaTGtG-3′ DMF17.7% 10 5′-AM-C6-atGTtcGGcaTGtG-3′ iBu  0.0% 115′-AM-C6-GGatGGtcGTaaGG-3′ DMF 21.7% 12 5′-AM-C6-GGatGGtcGTaaGG-3′ iBu 0.0% 13 5′-atGTt^(AMC6)cGGcaTGtG-3 DMF 25.0% 145′-atGTt^(AMC6)cGGcaTGtG-3′ iBu  0.0% 15 5′-AM-TEG-GGatGGtcGTaaGG-3′ DMF19.6% 16 5′-AM-TEG-GGatGGtcGTaaGG-3′ iBu  0.0% 175′-GGatGGtcGTaaGG-AM-C7-3′ DMF 13.4% 18 5′-GGatGGtcGTaaGG-AM-C7-3′ iBu 0.0% Upper case = LNA, lower case = DNA. AM-C6 = 6-aminohexyl, t^(AMC6)= 5-[N-(aminohexyl)-3-acrylimido]-2′-deoxyuridine, AM-TEG =11-amino-3,6,9-trioxaundecan-1-yl, AM-C7 =6-amino-2-(hydroxymethyl)-hexyl. The examples were fullyphosphorothioate oligonucleotides, where the LNA monomers werebeta-D-oxy LNA. The LNA-C monomers are 5-methyl cytosine LNA monomers.

Example 2

A 5′-aminohexyl linked phosphorthioate oligonucleotide with sequence5′-AM-C6-caGCGtaaagagAGG-3′ was prepared using DMF-protected LNA-G andiBu-protected DNA-G phosphoramidites as described in example 1. Thecrude material contained the full length product (FL) and approximately20% of the formyl (HCO, +28) impurity (FL+28)

By analysis with an ultra-high resolution mass spectrometer (FT-ICR-MStype; Thermo LTQ-FT Ultra) we determined the exact mass difference ofthe two [M-5H]⁵ peaks to 27.9968 Da (for FL 1044.11945 Da, for FL+281049.71881 Da) (FIG. 1) The mass difference of 5.59936 Da for the5-times charged ions results in 5.59936 Da*5=27.9968 Da for theuncharged molecule (FIG. 3).

The measured mass is within Δ=1.885 mmu accuracy to a carbonyl group(CO) and the potential next modification with a nominal mass of +28 Dais “N2” where the exact mass difference is −9.348 mmu which is alreadyoutside of the mass accuracy of the instrument used.

For determination of the exact position of the CO-modification a massspectrometric sequencing of the molecule was done by MS/MS with the sameinstrument. For nomenclature of oligonuclteotide fragmentation seeMcLuckey et al, J. Am. Chem. Soc., 115, 25, 12085-12095.

In the MS/MS experiment both 5-time charged ions (w/ and w/omodification) were fragmented. Here we located the modification on ionsfrom the 5′-end up to the eighth nucleotide whereas the nucleotides fromthe 3′-end broke up at the same position with no modification. Nofurther 3′-fragments with longer sequences were detectable. In theresulting spectra the b4-ion (FIG. 4) was observed for FL and also forFL+28. A further fragmentation with MS3 of the b4-ion (FIG. 4) resultedin spectra which show amongst other signals the a2-B ion and theb4-Cytosine ion at the first nucleotide (b4-C). This two ions lead tothe proposal that the modification is located on the C6-aminolinker(FIG. 5).

Since only the amino linker group is present in both structures aspossible reaction partner for modification we conclude that a formylamide is formed at that position (FIG. 6).

1. A method of preparing a LNA oligonucleotide comprising the steps of:a) Incorporating at least one LNA-G monomer comprising an acyl protectedexocyclic nitrogen into an oligonucleotide b) Incorporating at least oneoptionally protected aliphatic amine group into the oligonucleotide c)deprotecting the acyl protected exocyclic nitrogen of the at least oneLNA-G monomer by removal of the acyl protection group. wherein steps a)and b) can occur in either order.
 2. The method according to claim 1wherein the optionally protected aliphatic amine group is a primary orsecondary amine.
 3. The method according to any one of claim 1 or 2,wherein the optionally protected aliphatic amine group is a nonnucleosidic amine group.
 4. The method according to any one of claims1-3, wherein the optionally aliphatic amine group is selected from thegroup consisting of an amino alkyl, alkylamino alkyl, piperidine,piperazine, pyrrolidine, & imidazole.
 5. The method according to any oneof claims 1-3, wherein the optionally aliphatic amine group is selectedfrom the group consisting of 5′-TFA-Amino-Modifier-C5-CEPhosphoramidite, 5′-TFA-Amino-Modifier C6-CE Phosphoramidite,11-(trifluoroacetamido)-3,6,9-trioxaundecan-1-yl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5′-TFA-Amino-Modifier-C12-CEPhosphoramidite, Amino-Modifier C2-dT-CE Phosphoramidite, Amino-ModifierC6-dA-CE Phosphoramidite, Amino-Modifier C6-dA-CE Phosphoramidite,Amino-Modifier C6-dT-CE Phosphoramidite, N2-Amino-Modifier C6 dG, FmocAmino-Modifier C6 dT, 3′-Amino-Modifier C7 CPG 1000, 3′-Amino-ModifierC6-dC CPG, 3′-Amino-Modifier C6-dC CPG, 3′-PT-Amino-Modifier C6 CPG,3′-Amino-Modifier C6-dT CPG, PC 5′-Amino-Modifier-CE Phosphoramidite,5′-Amino-Modifier C6-PDA, 5′-Amino-Modifier C12-PDA, 5′-Amino-ModifierTEG PDA, Amino-Modifier Serinol, & 3′-Amino-Modifier Serinol CPG.
 6. Themethod according to any one of claims 1-3, wherein the optionallyprotected aliphatic amine group is an amino hexyl linker.
 7. The methodaccording to any one of claims 1-6, wherein the aliphatic amine group isincorporated into the oligonucleotide via the incorporation of anamino-modified monomer.
 8. The method according to claim 7, wherein thealiphatic amino-modified monomer is a phosphoramidite, a H phosphonateor a phosphotriester monomer.
 9. The method according to claim 7,wherein the amino-modified monomer is a phosphoramidite.
 10. The methodaccording to any one of claims 1-9, wherein the acyl protection group onthe exocyclic nitrogen of the LNA-G monomer(s) consists or comprises agroup selected from the group consisting of an optionally substitutedalkyl-, alkenyl-, alkynyl-, cycloalkyl- or aryl-group, preferably froman optionally substituted C₁₋₆-alkyl-, C₂₋₆-alkenyl-, C₂₋₆-alkinyl-,C₃₋₇-cycloalkyl- or phenyl-group; wherein when substituted, the R groupmay be mono or poly substituted, e.g. with one or more substituentsselected from the group consisting of halogen, C₁₋₆-alkyl, C₂₋₆-alkenyl,C_(m)-alkynyl, C₁₋₆-alkoxy, optionally substituted aryloxy or optionallysubstituted aryl.
 11. The method according to any one of claims 1-10,wherein the acyl protection group on the exocyclic nitrogen of the LNA-Gmonomer(s) is selected from the group consisting of Isobuturyl (iBu),Acetyl (Ac), Phenoxyacetyl (PAC), p-Isopropylphenoxyacetyl (iPrPAC),phenylacetyl, Isopropyloxyacetyl, methoxyacetyl, benzoyl,p-methoxyphenylacetyl, diphenylacetyl, cyclohexylcarbonyl,1,1-dimethylpropanoyl, and p-tert-Butyl-phenoxyacetyl.
 12. The methodaccording to any one of claims 1-10, wherein the acyl protection groupon the exocyclic nitrogen of the LNA-G monomer(s) is selected from thegroup consisting of Isobuturyl (iBu), Acetyl (Ac), Phenoxyacetyl (PAC),& p-Isopropylphenoxyacetyl (iPrPAC).
 13. The method according to any oneof claims 1-12, wherein, if present, other G residues incorporated intothe oligonucleotide also comprise an acyl protection group, such as theacyl protection groups of any one of claims 10-12.
 14. The methodaccording to any one of claims 1-13, wherein the LNA-G monomer(s), andoptionally when present other G monomers, is a phosphoramidite, aH-phosphonate or a phosphotriester monomer.
 15. The method according toany one of claims 1-14, wherein the LNA-G monomer(s), and optionallywhen present other G monomers, is a phosphoramidite.
 16. The methodaccording to any one of claims 1-15 wherein the LNA-G monomer comprisesa 2′-O—CH2-4′ biradical in the furanose ring.
 17. The method accordingto any one of claims 1-16, wherein step c) further comprisesdeprotection of the primary amine group.
 18. The method according to anyone of claims 1-17, wherein step c) comprises deprotection of theoligonucleotide is performed in the presence of ammonia, such as using asolution comprising ammonium hydroxide.
 19. The method according to anyone of claims 1-18, wherein step c) is followed by an additional step(d) which comprises incorporating a conjugate group onto the aliphaticprimary amine group.
 20. The method according to claim 19, wherein theconjugate group is a non-nucleotide moiety, selected from the groupconsisting of a lipid, a sterol, a carbohydrate, a peptide and aprotein.
 21. The method according to any one of claims 1-20, wherein atleast steps a)-c) are performed on a solid support and are followed bythe cleavage of the oligonucleotide from the solid support which may beperformed during step c) or subsequent to step c).
 22. The methodaccording to any one of claims 1-21, wherein the acyl protectiongroup(s) is isobuturyl and the aliphatic primary amine group(s) is anaminohexyl linker.
 23. An LNA oligonucleotide which comprises at leastone LNA-G monomer comprising an acyl protected exocyclic nitrogen and atleast one optionally protected aliphatic amine group, wherein said LNAoligonucleotide is attached to a solid support.
 24. A pharmaceuticalcomposition comprising an LNA oligomer conjugate which comprises anLNA-G monomer and an aliphatic amine linker positioned between the 5′nucleotide of LNA oligomer and a conjugate moiety, and apharmaceutically acceptable diluent, carrier or adjuvant, wherein saidcomposition is essentially free of +28 adduct.
 25. Use of an LNA-Gmonomer comprising an acyl protected exocyclic nitrogen for use in thesynthesis of an aliphatic amine containing LNA oligonucleotide.
 26. Useof an LNA-G monomer comprising an acyl protected exocyclic nitrogen foruse in the synthesis of an aliphatic amine containing LNAoligonucleotide conjugate.